Compositions and methods for modulating inflammatory and degenerative disorder

ABSTRACT

The disclosure provides compounds and method useful for modulating gp130 biological activity. The disclosure also provides methods and compositions for treating disease and disorders associated with gp130 activity, particularly those associated with inflammation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/898,236, filed Jun. 10, 2020, which application is a continuation ofInternational Application Serial No. PCT/US2019/020058, filed Feb. 28,2019, which application claims priority under 35 U.S.C. § 119 fromProvisional Application Ser. No. 62/636,325, filed Feb. 28, 2018, thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The disclosure provides compounds and method useful for modulating gp130biological activity. The disclosure also provides methods andcompositions for treating disease and disorders associated with gp130activity, particularly those associated with inflammation.

BACKGROUND

Osteoarthritis (OA) is a degenerative disease of joints, characterizedby progressive loss of cartilage which causes stiffness, swelling andpain. Nearly 10% of the world's population suffers from OA, making itthe most common form of arthritis and one of the most commonpathological conditions. In the United States, there are over 27 millionpeople affected by OA, with this number projected to climb steeply dueto a rapidly aging population and increases in obesity rates. Each year,over $185 billion is spent to treat OA globally, establishing thisdisease as a major burden on global health and economics.

Currently, there are no disease modifying agents available on the marketfor the treatment of osteoarthritis. Treatment modalities are focused onlifestyle modifications, pain management and improving joint viscosity,with the overall goal to delay joint replacement surgery. Initialtherapies for those with mild osteoarthritis include weight loss,physical therapy and pain management using over-the-counternon-steroidal anti-inflammatory drugs (NSAIDs). As the conditionprogresses to a moderate stage, opioid-based pain control is introduced,while continuing physical therapy and other exercises. If the diseaseprogresses to a severe stage, intra-articular injections, such ashyaluronic acid, are used to increase joint viscosity. Finally, if noneof the previous treatments are able to mitigate pain, total jointreplacement surgery is considered. There are over 1 million knee and hipreplacements performed each year in the U.S, at a cost of over $50billion.

These glaring statistics, and the lack of effective treatments thataddress the causes rather than the symptoms of OA, have not escapednotice by clinicians and researchers. The most effective treatments forcartilage lesions thus far include four main surgical procedures:microfracture, osteochondral autologous transplantation (OATS),autologous chondrocyte implantation (ACI) and fresh osteochondralallograft (FOA). Each of these procedures has major disadvantages thatpreclude widespread adoption and implementation. Microfracture istechnically easy, but is prone to subsequent degeneration in comparisonto the other methods, and mostly results in the formation ofbiomechanically inferior fibrocartilage. OATS is technically difficult,and donor site morbidity increases with the number and size of theosteochondral plugs that are removed for transplantation. ACI is by farthe most expensive cartilage defect treatment, and recovery biopsiesstill display fibrocartilaginous content in place of hyaline cartilage.FOA is also expensive due to specific instrumentation required for theprocedure, limited availability of tissue and short shelf-life ofmaterial for transplantation (less than 6 weeks). Critically, all ofthese techniques require surgery in order to be implemented; this fact,in combination with the unconvincing data surrounding their efficacy,has limited the broad implementation of these therapies.

Newer efforts in cell therapy are focusing on transplantation ofchondrocytes from other sources or mesenchymal stem cells into thejoint. Many groups are optimizing strategies to generate articular-likechondrocytes from pluripotent stem cells for use in cartilage repair.This approach, although promising, will face many of the same hurdlesthat impede the adoption of existing cell therapies: integration oftransplanted cells with existing cartilage, inflammation and graftsurvival. Other groups are pursuing a strategy similar to microfracture,in that they are attempting to promote the adoption of an articularcartilage fate by mesenchymal stem cells isolated from a variety ofsources. Because the starting material for these procedures can beexpanded in culture and applied allogeneically, several groups havereached Phase 2 clinical trials with their work in this area. However,as with microfracture, concerns over the ability of the cells togenerate true articular cartilage (vs. fibrocartilage) thatsubstantially integrates with surrounding tissue loom.

SUMMARY

The disclosure provides a compound comprising the structure of FormulaI, II or III:

wherein,

-   -   X is:

-   -   Y is:

-   -   Z is:

X¹ is S, CH, or NH;X² is S, CH, or N;X³ is CR² or S;Y¹ is CR⁷ or N;Y² is CR⁶ or N;Y³ is CR⁵ or N;Z¹ is O or CH;Z² is O, N, NH or CH;Z³ is CR⁹ or N;Z⁴ is CR⁸ or N;Z⁵ is N or CR¹⁴;Z⁶ is N or CR¹³;Z⁷ is N or CR¹²;Z⁸ is N or CR¹¹;Z⁹ is N or CR¹⁰;v is 0 or 1;R¹, R², R⁸ and R⁹ are independently selected from H, D, (C₁-C₃) alkyl,(C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro, thiol, and amino;R³-R⁷ and R¹⁰-R¹⁴ are independently selected from H, D, (C₁-C₃) alkyl,(C₁-C₃) alkenyl, halo, cyano, hydroxyl, nitro, thiol, amino, OC (R¹⁵)₃,OCH(R¹⁵)₂, OCH2(R¹⁵),

wherein n is an integer from 1 to 5; andR¹⁵ is independently H, halo, or a (C₁-C₃) alkyl;

-   -   and wherein the compound interacts with domain 2 of gp130 and        which locks gp130 into a non-permissive conformation and/or        produce atypical gp130 homodimers. In one embodiment, X is        selected from the group consisting of

and wherein R² is an H, D, (C¹-C₃)alkyl, (C₁-C₃) alkenyl, halo, cyano,hydroxyl, nitro, thiol, or amino. In another or further embodiment, Y isselected from the group consisting of:

and wherein v is 0 or 1; R³-R⁷ are independently selected from H, D,(C₁-C₃)alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro, thiol,amino, OC(R¹⁵)₃, OCH(R¹⁵)₂, OCH2(R¹⁵),

and wherein n is an integer from 1 to 5. In still another of furtherembodiment of any of the foregoing, Z is selected from the groupconsisting of:

and wherein, R⁸ and R⁹ are independently selected from H, D, (C₁-C₃)alkyl, (C₁-C₃) alkenyl, halo, cyano, hydroxyl, nitro, thiol, and amino;R¹⁰-R¹⁴ are independently selected from H, D, (C₁-C₃) alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro, thiol, amino, OC(R¹⁵)₃,OCH(R¹⁵)₂, OCH₂(R¹⁵),

wherein n is an integer from 1 to 5; and wherein R¹⁵ is independently H,halo, or a (C₁-C₃) alkyl. In still another or further embodiment of anyof the foregoing the compound has a structure selected from the groupconsisting of:

In another embodiment, the compound modulates STAT3 and MYC signaling.In one embodiment, the compound does not stimulate STAT3 and MYCsignaling. In another embodiment, the compound stimulates STAT3 and MYCsignaling.

The disclosure also provides a method of treating an inflammatorydisease or disorder or cell-proliferative disease or disorder comprisingcontacting a subject with a compound as described herein and above. Inone embodiment, the inflammatory disease or disorder or cellproliferative disease or disorder is selected from the group consistingof stroke; heart disease; cartilage degeneration; hair loss; woundhealing; arthritis; neurodegenerative disorders; aging; diseases knownto be associated with low grade chronic inflammation; immune disordersincluding psoriasis, rosacea, lupus, rheumatoid arthritis, inflammatorybowel disease; and cancer.

The disclosure also provides a method of modulating IL-6 mediatedinflammatory responses in a cell comprising contacting the cell with acompound as described herein and above. In one embodiment, the cell is achondrocyte.

The disclosure also provides a composition comprising a pharmaceuticallyacceptable carrier and a compound as described herein and above.

The disclosure also provides a method of treating an acute of chronicinflammatory state comprising contact a subject with a compound orpharmaceutical as described herein and above.

The disclosure also provides a method of decreasing an activatedinflammatory pathway in a cell comprising contact the cell with acompound or pharmaceutical as described herein and above.

The disclosure provides a method of inhibiting the production orinduction of pro-inflammatory genes, cytokines or mediators comprisingcontacting a cell or subject with a compound or pharmaceutical asdescribed herein and above.

The disclosure provides a method of inhibiting the production orinduction of extracellular matrix degrading enzymes comprisingcontacting a cell or subject with a compound or pharmaceutical asdescribed herein and above.

DESCRIPTION OF DRAWINGS

FIG. 1A-B shows (A) Expression of gp130 is enriched on superficialchondrocytes that are also marked by BMPR1B in human adolescents (lefttwo columns) and (B) adults.

FIG. 2A-D shows (A) A screen of the ability of 170,000 compounds toinhibit a Co110a1-RFP reporter in limb bud mesenchymal cells identifiedRCGD 423 (B-C). The ability of the compound to inhibit hypertrophy wasconfirmed on human fetal articular chondrocytes (D).

FIG. 3 shows a schematic of molecular and functional outcomes downstreamof pro-inflammatory cytokines.

FIG. 4A-B shows IL-6 family cytokines activate the MAPK, AKT and NF-κBpathways in adult pig articular chondrocytes; RCGD 423 does not, despiteshared increase in active STAT3 (pSTAT3) and MYC proteins.

FIG. 5A-B shows (A) IL-6 family cytokines increase the expression ofproteolytic enzymes while RCGD 423 does not. (B) Accumulation of pSTAT3and MYC proteins induced by RCGD 423 can be blocked by chemicalinhibition of JAK or gp130 proteins.

FIG. 6 shows molecular modeling of the proposed interaction betweengp130 and RCGD 423. Oxygen, nitrogen; sulfur and bromine atom are shown.

FIG. 7A-B shows gp130^(−/−) Ba/F3 cells (A) only increase gp130signaling in the presence of RCGD 423 and WT gp130 (B).

FIG. 8 shows Injection of RCGD 423 into rat joints increased pSTAT3 andMYC levels; this could be blocked by co-injection of the small moleculegp130 inhibitor SC144.

FIG. 9 shows Tagged versions of gp130 allow interrogation of hetero- vs.homodimeric signaling. gp130-Myc was only detectable in the presence ofRCGD 423.

FIG. 10A-B shows RCGD 423 reduces signaling downstream of OSM and IL-6(A) by competing for gp130 (B).

FIG. 11 shows RCGD 423 directly competes with OSMR and IL-6R for gp130occupancy in chondrocytes. Adult human chondrocytes transfected withgp130-Flag were incubated with various concentrations of RCGD 423 in thepresence of IL-6 or OSM; levels of immoprecipitated IL-6R or OSMR wereused to calculate the dissociation constant (K_(d)).

FIG. 12A-E shows A rat model of OA was validated (A and B) to assess theability of RCGD 423 to prevent loss of Safranin O⁺ hyaline cartilage (C)and joint degeneration (D); the drug also decreased the levels ofcatabolic enzymes (E).

FIG. 13 shows Treatment of rats following partial meniscectomy with RCGD423 reduced the size and maturity of osteophytes (arrow).

FIG. 14A-B shows RCGD 423 inhibits increases in ERK and NF-κB levelsdownstream of OSM and IL-6 in adult human (A) synoviocytes and (B)PBMCs.

FIG. 15A-C shows CX-011 has little effect on endogenous levels ofproteins mediating pro-inflammatory signals but strongly inhibitsincreases in these proteins in the presence of OSM (A). CX-011 preventsthe breakdown of collagen and aggrecan in pig articular cartilageexplants driven by OSM as indicated by lower levels of degenerationepitopes (B) and increase in catalytic enzymes (C).

FIG. 16A-C shows Identification of novel prospective small moleculemodulators of gp130 receptor. (A) Class 1 molecules (marked by redarrows) activate STAT3 and MYC signaling pathways wile Class 2 molecules(blue arrows) have no stimulatory effects on STAT3 and MYC signaling inchondrocytes as determined by western blot analysis. (B) Structures ofthe most prominent Class 1 and Class 2 molecules. (C) Shows additionalmolecules tested for effects on STAT and MYC signaling. All drugs weretested at 10 μM and assessed using Western Blot.

FIG. 17 shows Class 1 (red ovals) and Class 2 (blue ovals) moleculesdramatically inhibit Inflammation-induced degradation of cartilagematrix proteins, as determined by measuring the levels MMP13 andADAMTS4. Please note that pro-inflammatory cytokine oncostatin M (OSM)upregulates levels of MMP13 and ADAMTS proteases while in the presenceof selected class 1 and 2 molecules this effect is mitigated.

FIG. 18 shows possible binding mode for designed molecule B8 with GP130D2 Primarily the interactions are hydrophobic in nature. The designedmolecule1 is nicely fit in to the hydrophobic packet of the GP130 likeRGD423. The residues interacting with the designed molecule1 are shownin stick representation. Fitness score: −1755.2 and DeltaG: −7.71kcal/mol. SwissDoc Software.

FIG. 19A-B shows that different test chemicals act as activators,neutral effectors or inhibitors for the expression of pSTAT3 and MYC incultured porcine chondrocytes. All drugs tested at 10 μM and assessedusing Western Blot.

FIG. 20 shows that several analogs (e.g., B8, 803, 805 815) inhibitOSM-mediated activation of gp130. 423F is the positive control drug.Adult human articular chondrocytes from three independent donors wereused to generate the results.

FIG. 21 shows that 803 and 815 inhibit OSM-mediate d activation ofgp130. Adult human articular chondrocytes from three independent donorswere used to generate the results.

FIG. 22A-C show matrix degradation assays. Several analogs (e.g., 805,839) strongly prevent the breakdown of collagen and aggrecan in pigarticular cartilage explants driven by OSM as indicated by lower levelsof degeneration epitopes for aggrecan (aggrecan-neo) and collegen 2(collagen-neo).

FIG. 23 shows that some pSTAT3 and MYC activating analogs (e.g., 810,840) can also strongly prevent the breakdown of collagent and aggrecanin pig articular cartilage explants driven by OSM as indicated by lowerlevels of degeneration epitopes for aggrecan (aggrecan-neo) and collagen2 (collagen-neo).

FIG. 24 shows that some analogs (e.g., 826, 843, 849) can still stronglyprevent the breakdown of collagen and aggrecan in pig articularcartilage explants driven by OSM as indicated by lower levels ofdegeneration epitopes for aggrecan (aggrecan-neo) and collagen 2(collagen-neo).

FIG. 25A-B shows that molecule 805 strongly inhibits OSM-mediatedactivation of gp130 (A) and its downstream effector JAK2 (B). Adulthuman articular chondrocytes from three independent donors were used togenerate these results.

FIG. 26 shows validations of the effect of molecule 805 on fresh pigcartilage explants. 72 hour exposure to the drug and OSM. 805 stronglyprevents the breakdown of collagen and aggrecan in pig articularcartilage explants driven by OSM as indicated by lower levels ofdegeneration epitopes for aggrecan (aggrecan-neo) and collagen 2(collagen-neo). Effect is concentration dependent and increases withincrease in drug concentration.

FIG. 27A-C shows 805 has little effect on endogenous levels of proteinsmediating pro-inflammatory signals but strongly inhibits increases inthese proteins in the presence of OSM (A). 805 prevents the breakdown ofcollagen and aggrecan in pig articular cartilage explants driven by OSMas indicated by lower levels of degeneration epitopes (B) and increasein catalytic enzymes (C).

FIG. 28A-C shows 805 inhibits inflammatory response in vivo in adose-dependent manner. Rat (Sprague-Dawley wild-type) were injected withComplete Freund's Adjuvant (CFA) to induce systemic inflammatoryresponse. Drug (805) was injected intra-articularly; multiple doses (Aand B, N=3). 805 showed prolonged effect preventing activation gp130 insick animals as well as leading to lower activity of inflammatoryindicators such as NF-kB and pSRC kinase.

FIG. 29 shows a receptor competition assay. Adult human chondrocytes;IP-FLAG AB (transfected with gp130 FLAG plasmid), WB-OSMR. This assayshows that 805 disrupts the interaction between OSMR and gp130 in aconcentration-dependent manner.

FIG. 30 shows that molecule 826 strongly inhibits OSM-mediatedactivation of gp130 and downstream effectors of chronic inflammationincluding JAK2, SHP2, NF-kB, and SRC. Adult human articular chondrocytesfrom three independent donors were used.

FIG. 31 shows molecules of the disclosure inhibit OSM-induced NF-kBactivation in BaF3 via transgenic gp130. BaF3 cells were transfectedwith WT gp130 plasmid.

FIG. 32 shows molecules 805 and 826 strongly inhibit OSM-mediatedactivation of gp130 and downstream effectors of chronic inflammationincluding NF-kB in human immune cells. Cells from three independentdonors were used to generate the results.

FIG. 33A-B shows that molecule 805 strongly inhibits OSM-mediatedsecretion of TNF-α (A) and IL-6 (B). Adult synovial cells from threeindependent donors were used. Molecule 805 was used at 10 μM.

FIG. 34 shows that oral administration of a single dose of molecule 805reduced synovial inflammation in rat joints.

FIG. 35 shows that intra-articular injection of molecule 805 reducescartilage degeneration following medial meniscal tear surgery.

FIG. 36 shows a graph of pain assessment (spontaneous activity) inCFA-induced arthritis. Number of runs per hour was determined using awell-accepted activity wheel system installed in housing cages andequipped with an automatic counter. Control rats were treated withvehicle, molecule 805 was injected systemically (10 mg/kg) for 15 days.

FIG. 37 shows survival analysis of NZM.2328 female mice, whichspontaneously developed a lethal lupus nephritis-like syndrome with 90%penetrance survive longer when injected intraperitoneally twice weeklywith 10 mg/kg of molecule 805.

FIG. 38 are blots from NZM.2328 mice that spontaneously developed severeimmune inflammatory disease similar to human lupus. Incubation of spleencells obtained from these mice with molecules 805 and 839 results insignificant reduction of inflammatory activity (marked with NF-kB) inthese cells compared to control cells treated with DMSO.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a chondrocyte” includes aplurality of such chondrocytes and reference to “an antagonist” includesreference to one or more antagonists or equivalents thereof known tothose skilled in the art, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although many methods andreagents similar to or equivalent to those described herein can be usedin the practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior disclosure.

The term “alkyl” refers to an organic group that is comprised of carbonand hydrogen atoms that contains single covalent bonds between carbons.Typically, an “alkyl” as used in this disclosure, refers to an organicgroup that contains 1 to 30 carbon atoms, unless stated otherwise. Whereif there is more than 1 carbon, the carbons may be connected in a linearmanner, or alternatively if there are more than 2 carbons then thecarbons may also be linked in a branched fashion so that the parentchain contains one or more secondary, tertiary, or quaternary carbons.An alkyl may be substituted or unsubstituted, unless stated otherwise.

The term “alkenyl”, refers to an organic group that is comprised ofcarbon and hydrogen atoms that contains at least one double covalentbond between two carbons. Typically, an “alkenyl” as used in thisdisclosure, refers to organic group that contains 2 to 30 carbon atoms,unless stated otherwise. While a C₂-alkenyl can form a double bond to acarbon of a parent chain, an alkenyl group of three or more carbons cancontain more than one double bond. It certain instances the alkenylgroup will be conjugated, in other cases an alkenyl group will not beconjugated, and yet other cases the alkenyl group may have stretches ofconjugation and stretches of non-conjugation. Additionally, if there ismore than 2 carbon, the carbons may be connected in a linear manner, oralternatively if there are more than 3 carbons then the carbons may alsobe linked in a branched fashion so that the parent chain contains one ormore secondary, tertiary, or quaternary carbons. An alkenyl may besubstituted or unsubstituted, unless stated otherwise.

The term “alkynyl”, refers to an organic group that is comprised ofcarbon and hydrogen atoms that contains a triple covalent bond betweentwo carbons. Typically, an “alkynyl” as used in this disclosure, refersto organic group that contains 2 to 30 carbon atoms, unless statedotherwise. While a C₂-alkynyl can form a triple bond to a carbon of aparent chain, an alkynyl group of three or more carbons can contain morethan one triple bond. Where if there is more than 2 carbon, the carbonsmay be connected in a linear manner, or alternatively if there are morethan 4 carbons then the carbons may also be linked in a branched fashionso that the parent chain contains one or more secondary, tertiary, orquaternary carbons. An alkynyl may be substituted or unsubstituted,unless stated otherwise.

The term “aryl”, refers to a conjugated planar ring system withdelocalized pi electron clouds that contain only carbon as ring atoms.An “aryl” for the purposes of this disclosure encompasses from 1 to 7aryl rings wherein when the aryl is greater than 1 ring the aryl ringsare joined so that they are linked, fused, or a combination thereof. Anaryl may be substituted or unsubstituted, or in the case of more thanone aryl ring, one or more rings may be unsubstituted, one or more ringsmay be substituted, or a combination thereof. More specifically,substituted aryl groups include acetylphenyl groups, particularly4-acetylphenyl groups; fluorophenyl groups, particularly 3-fluorophenyland 4-fluorophenyl groups; chlorophenyl groups, particularly3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups,particularly 4-methylphenyl groups, and methoxyphenyl groups,particularly 4-methoxyphenyl groups.

The “binding site 1” of the gp130 receptor, or “binding site 1 gp 130receptor” is a binding pocket within the gp 130 receptor. The sequencefor the gp130 receptor is set forth in SEQ ID NO: 1. The sequence forthe binding site 1 of the gp130 receptor is set forth in SEQ ID NO:2 andSEQ ID NO:3 and includes amino acids corresponding to positions 173,174, 175 and 176 in gp130 or amino acid residues KAKR as set forth inSEQ ID NO:4. An amino acid residue in a protein or receptor“corresponds” to a given residue when it occupies the same essentialstructural position within the protein or receptor as the given residue,for example, in homologous proteins that may have a different numberingconvention. The amino acid residues and fragments of gp130 disclosedherein are referred to as corresponding to the entire (918 amino acid)length of gp130 and/or gp130 without the signaling fragment that is 22amino acid residues in length (e.g, KAKR as set forth in SEQ ID NO:4 isreferred to herein as corresponding to positions 173, 174, 175 and 176and/or positions 151, 152, 153 and 154 in gp130).

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “cycloalkyl”, as used in this disclosure, refers to an alkylthat contains at least 3 carbon atoms but no more than 12 carbon atomsconnected so that it forms a ring. A “cycloalkyl” for the purposes ofthis disclosure encompass from 1 to 7 cycloalkyl rings, wherein when thecycloalkyl is greater than 1 ring, then the cycloalkyl rings are joinedso that they are linked, fused, or a combination thereof. A cycloalkylmay be substituted or unsubstituted, or in the case of more than onecycloalkyl ring, one or more rings may be unsubstituted, one or morerings may be substituted, or a combination thereof.

The term “cycloalkenyl”, as used in this disclosure, refers to an alkenethat contains at least 3 carbon atoms but no more than 12 carbon atomsconnected so that it forms a ring. A “cycloalkenyl” for the purposes ofthis disclosure encompass from 1 to 7 cycloalkenyl rings, wherein whenthe cycloalkenyl is greater than 1 ring, then the cycloalkenyl rings arejoined so that they are linked, fused, or a combination thereof. Acycloalkenyl may be substituted or unsubstituted, or in the case of morethan one cycloalkenyl ring, one or more rings may be unsubstituted, oneor more rings may be substituted, or a combination thereof.

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with the compounds/moleculesor methods provided herein. Disease as used herein may refer toinflammatory diseases and disorders and immune diseases and disorderssuch as cartilage degenerative disease, joint surface injury orarthritis (including rheumatoid arthritis), psoriasis, inflammatorybowel disease, aging, lupus, rosacea and the like.

For purposes of this disclosure, the term “extended mixed ring system”refers to a group that is comprised of at least 2 ring structures, butno more than 7 ring structures. An “extended mixed ring system” iscomprised of at least one ring functional group that is different fromanother ring functional group. Examples of ring groups include, but arenot limited to, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, andheterocycle. Each ring may be optionally substituted. The ringscomprising the mixed extended ring system may be joined so that they arelinked, fused, or a combination thereof.

The term “functional group” or “FG” refers to specific groups of atomswithin molecules that are responsible for the characteristic chemicalreactions of those molecules. While the same functional group willundergo the same or similar chemical reaction(s) regardless of the sizeof the molecule it is a part of, its relative reactivity can be modifiedby nearby functional groups. The atoms of functional groups are linkedto each other and to the rest of the molecule by covalent bonds.Examples of FG that can be used in this disclosure, include, but are notlimited to, substituted or unsubstituted alkyls, substituted orunsubstituted alkenyls, substituted or unsubstituted alkynyls,substituted or unsubstituted aryls, substituted or unsubstitutedhetero-alkyls, substituted or unsubstituted hetero-alkenyls, substitutedor unsubstituted hetero-alkynyls, substituted or unsubstitutedcycloalkyls, substituted or unsubstituted cycloalkenyls, substituted orunsubstituted hetero-aryls, substituted or unsubstituted heterocycles,halos, hydroxyls, anhydrides, carbonyls, carboxyls, carbonates,carboxylates, aldehydes, haloformyls, esters, hydroperoxy, peroxy,ethers, orthoesters, carboxamides, amines, imines, imides, azides, azos,cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosos,nitros, nitrosooxy, pyridyls, sulfhydryls, sulfides, disulfides,sulfinyls, sulfos, thiocyanates, isothiocyanates, carbonothioyls,phosphinos, phosphonos, phosphates, Si(OH)₃, Ge(OH)₃, Sn(OH)₃, Si(SH)₄,Ge(SH)₄, AsO₃H, AsO₄H, P(SH)₃, As(SH)₃, SO₃H, Si(OH)₃, Ge(OH)₃, Sn(OH)₃,Si(SH)₄, Ge(SH)₄, Sn(SH)₄, AsO₃H, AsO₄H, P(SH)₃, and As(SH)₃.

The term “gp130” as used herein refers to glycoprotein 130, a cellsurface receptor that is expressed ubiquitously in the body. Activationof gp130 is essential for several physiological functions, including butnot limited to, acute-phase response to injury and infection, fertility,metabolism, haematopoiesis, neuroprotection, anti-angiogenesis, andmelanoma and tumor cell suppression. Gp130 is activated by a ligand fromthe IL-6 family of cytokines, including but not limited to, IL-6, IL-11,leukemia inhibitory factor (LIF), Oncostatin M (OSM), ciliaryneurotrophic factor (CNTF), cardiotrophin-1 (CT-1) andcardiotrophin-like cytokine (CLC). Activation of gp130 signaling may bedirect, i.e. activation may be triggered by binding of the liganddirectly to gp130 (i.e., IL-6 or IL-11, which result ingp130-homodimerization). Activation of gp130 signaling may also beindirect by binding of the ligand to another cell surface receptor,which forms a complex with gp130, thereby activating it. LIF, CT-1,CNTF, OSM and CLC form heterodimers of gp130 and LIFR, whereas OSM mayalso form a heterodimer of gp130 and OSMR. Therefore, LIF, CT-1, CNTF,OSM and CLC may activate gp130 signaling directly, by binding gp130first, or indirectly, by binding LIFR/OSMR and then recruiting gp130 tothe complex. The ligands of the IL-6 cytokine family trigger theJAK/STAT pathway, the first event of which is the ligand-induced homo-or hetero-dimerization of signal-transducing receptor subunits. AllIL-6-type cytokines recruit gp130 to their receptor complexes. Theyeither signal via gp130 alone or in combination with LIFR or OSMR, whichare all able to activate Jaks and to recruit STAT proteins. The terms“gp130 receptor,” “gp130,” gp130 protein,” “IL6ST receptor,” “IL6ST” or“IL6ST protein” are here used interchangeably and according to theircommon, ordinary meaning (e.g., transmembrane protein “glycoprotein130”) and refer to proteins of the same or similar names and functionalfragments and homologs thereof. The term includes any recombinant ornaturally occurring form of, or variants thereof that maintain gp130activity (e.g. within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or 100% activity compared to gp130). In embodiments, the gp 130 receptorhas at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO:1 or a functional fragment thereof (e.g. 700contiguous amino acids of SEQ ID NO:1, 750 contiguous amino acids of SEQID NO:1, 800 contiguous amino acids of SEQ ID NO:1, 850 contiguous aminoacids of SEQ ID NO:1 870 contiguous amino acids of SEQ ID NO:1, 880contiguous amino acids of SEQ ID NO:1, 890 contiguous amino acids of SEQID NO: 1, 900 contiguous amino acids of SEQ ID NO:1 or 910 contiguousamino acids of SEQ ID NO:1).

The term “hetero-” when used as a prefix, such as, hetero-alkyl,hetero-alkenyl, hetero-alkynyl, or hetero-hydrocarbon, for the purposeof this disclosure refers to the specified hydrocarbon having one ormore carbon atoms replaced by non-carbon atoms as part of the parentchain. Examples of such non-carbon atoms include, but are not limitedto, N, O, S, Si, Al, B, and P. If there is more than one non-carbon atomin the hetero-based parent chain then this atom may be the same elementor may be a combination of different elements, such as N and O.

The term “heterocycle”, as used in this disclosure, refers to ringstructures that contain at least 1 noncarbon ring atom. A “heterocycle”for the purposes of this disclosure encompass from 1 to 7 heterocyclerings wherein when the heterocycle is greater than 1 ring theheterocycle rings are joined so that they are linked, fused, or acombination thereof. A heterocycle may be a hetero-aryl or nonaromatic,or in the case of more than one heterocycle ring, one or more rings maybe nonaromatic, one or more rings may be hetero-aryls, or a combinationthereof. A heterocycle may be substituted or unsubstituted, or in thecase of more than one heterocycle ring one or more rings may beunsubstituted, one or more rings may be substituted, or a combinationthereof. Typically, the noncarbon ring atom is N, O, S, Si, Al, B, or P.In case where there is more than one noncarbon ring atom, thesenoncarbon ring atoms can either be the same element, or combination ofdifferent elements, such as N and O. Examples of heterocycles include,but are not limited to: a monocyclic heterocycle such as, aziridine,oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline,imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydro-pyridine, piperazine, morpholine,thiomorpholine, pyran, thiopyran, 2,3-dihydropyran, tetrahydropyran,1,4-dihydropyridine, 1,4-dioxane, 1,3-dioxane, dioxane, homopiperidine,2,3,4,7-tetrahydro-1H-azepine homopiperazine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethylene oxide; and polycyclicheterocycles such as, indole, indoline, isoindoline, quinoline,tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline,1,4-benzodioxan, coumarin, dihydrocoumarin, benzofuran,2,3-dihydrobenzofuran, isobenzofuran, chromene, chroman, isochroman,xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole,purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, phenanthridine, perimidine, phenanthroline, phenazine,phenothiazine, phenoxazine, 1,2-benzisoxazole, benzothiophene,benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine,carbazole, carboline, acridine, pyrolizidine, and quinolizidine. Inaddition to the polycyclic heterocycles described above, heterocycleincludes polycyclic heterocycles wherein the ring fusion between two ormore rings includes more than one bond common to both rings and morethan two atoms common to both rings. Examples of such bridgedheterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and7-oxabicyclo[2.2.1]heptane.

“Inflammation” refers to a complex biological response of a body to astimulus (e.g., a pathogen, cellular damage or an irritant).Inflammation, when prolonged, can lead to an inflammatory disease ordisorder. Factors elicited during an inflammatory reaction includepro-inflammatory cytokines (e.g., TNF-a, IL-1, INF-γ, MCP-1), cellularmigration (e.g., monocytes, macrophages, lymphocytes, plasma cells) andserum proteins (e.g., serum amyloid A (SAA) and serum amyloid P (SAP)).Inflammation can be local (e.g., vascular inflammation) or systemic.

“Inflammatory disorder” or “inflammatory disease” refers to a conditioncharacterized by inflammation in a cell, tissue or body. Inflammatorydiseases and disorders include, but are not limited to,hypersensitivities (e.g., allergies), asthma, autoimmune disease (e.g.,rheumatoid and osteo arthritis, lupus, multiple sclerosis), cancer,diabetes, inflammatory bowel disease (IBD) or cardiovascular disease(e.g., atherosclerosis), NAFLD, NASH, hepatitis, fibrosis, andcirrhosis.

The term “mixed ring system” refers to optionally substituted ringstructures that contain at least two rings, and wherein the rings arejoined together by linking, fusing, or a combination thereof. A mixedring system comprises a combination of different ring types, includingcycloalkyl, cycloalkenyl, aryl, and heterocycle.

The term “pharmaceutically acceptable” as in pharmaceutically acceptablesalt or pharmaceutically acceptable counter ion, refers to compounds,salts, or ions that are tolerated by a subject for topical, or internaluse.

The term “pharmaceutically acceptable salt” refers to making a saltformation of a compound disclosed herein. Salt formation can be used asa means of varying the properties of the compounds disclosed herein, forexample, to increase or decrease solubility of the compounds, to improvestability of the compounds, to reduce toxicity of the compounds, and/orto reduce the hygroscopicity of the compounds. There are a wide range ofchemically diverse acids and bases, with a range of pKa values,molecular weights, solubilities and other properties, that can used formaking pharmaceutically acceptable salts of the compounds disclosedherein. Examples of pharmaceutically acceptable acid addition saltsinclude, but are not limited to, hydrochloride, hydrobromide,hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, tartrate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucaronate, saccharate, formate, benzoate,glutamate, methanesulfonate, ethanesulfonate, benzensulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds ofthe disclosure can form pharmaceutically acceptable salts with variousamino acids. Examples of pharmaceutically acceptable base addition saltsinclude, but are not limited to, aluminum, calcium, lithium, magnesium,potassium, sodium, zinc, and diethanolamine salts. For additionalexamples of pharmaceutical salts that can used to practice thisdisclosure, see P. H. Stahl and C. G. Wermuth (eds.), PharmaceuticalSalts: Properties, Selection, and Use (2d ed. 2011) Wiley and SonsPublisher, ISBN: 978-3-90639-051-2.

The term “pharmaceutically acceptable counter ion” either refers topharmaceutically acceptable cations including, but not limited to,alkali metal cations (e.g., Li⁺, Na⁺, K⁺), alkaline earth metal cations(e.g., Ca²⁺, Mg²⁺), non-toxic heavy metal cations and ammonium (NH₄ ⁺)and substituted ammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl, orsubstituted alkyls, i.e., including, methyl, ethyl, or hydroxyethyl,specifically, trimethyl ammonium, triethyl ammonium, and triethanolammonium cations); or pharmaceutically-acceptable anions including, butnot limited to, halides (e.g., Cl⁻, Br⁻), sulfate, acetates (e.g.,acetate, trifluoroacetate), ascorbates, aspartates, benzoates, citrates,and lactate.

A “subject” generally refers to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals. Animals include all vertebrates,e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens,amphibians, and reptiles.

The term “substantially” as used to modify a term means that themodified term includes minor variations in size, purity, structure andthe like by only a minor amount. Accordingly, “substantially homogenousin size” means that the material does not vary by more than 1%, 5%, 10%,20% or 30% (or any value there between) in size from an average size.

The term “substituted” with respect to heterocycles, and the like,refers to structures wherein the parent chain contains one or moresubstituents.

The term “substituent” refers to an atom or group of atoms substitutedin place of a hydrogen atom. For purposes of this disclosure, asubstituent would include deuterium atoms.

A “therapeutically effective amount,” refers to an amount of a compound,molecule or composition of the disclosure that reduces a symptom orsymptoms (and grammatical equivalents of this phrase) or the severity ofor frequency of the symptom(s), or elimination of the symptom(s)associated with a disease or disorder to be treated. A “prophylacticallyeffective amount” of a drug is an amount of a drug that, whenadministered to a subject, will have the intended prophylactic effect,e.g., preventing or delaying the onset (or reoccurrence) of an injury,disease, pathology or condition, or reducing the likelihood of the onset(or reoccurrence) of an injury, disease, pathology, or condition, ortheir symptoms. The full prophylactic effect does not necessarily occurby administration of one dose, and may occur only after administrationof a series of doses. Thus, a prophylactically effective amount may beadministered in one or more administrations. The exact amounts willdepend on the purpose of the treatment, and will be ascertainable by oneskilled in the art using known techniques (see, e.g., Lieberman,Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Scienceand Technology of Pharmaceutical Compounding (1999); Pickar, DosageCalculations (1999); and Remington: The Science and Practice ofPharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams &Wilkins).

The term “unsubstituted” with respect to hydrocarbons, heterocycles, andthe like, refers to structures wherein the parent chain contains nosubstituents.

As used herein, a wavy line intersecting another line that is connectedto an atom indicates that this atom is covalently bonded to anotherentity that is present but not being depicted in the structure. A wavyline that does not intersect a line but is connected to an atomindicates that this atom is interacting with another atom by a bond orsome other type of identifiable association.

A bond indicated by a straight line and a dashed line indicates a bondthat may be a single covalent bond or alternatively a double covalentbond. But in the case where an atom's maximum valence would be exceededby forming a double covalent bond, then the bond would be a singlecovalent bond.

It should be understood many of the reagents and starting materials usedin the Schemes presented herein are readily available from variouscommercial suppliers, such as Sigma-Aldrich, Alfa Aesar, Tokyo ChemicalIndustry Co., LTD, etc. Moreover, many of these same reagents andstarting materials can be modified to incorporate additional functionalgroups by using standard organic synthesis reactions.

When a compound disclosed herein contains an acidic or basic moiety, itmay also disclosed as a pharmaceutically acceptable salt (See, Berge etal., J. Pharm. Sci. 1977, 66, 1-19; and “Handbook of PharmaceuticalSalts, Properties, and Use,” Stah and Wermuth, Ed.; Wiley-VCH and VHCA,Zurich, 2002).

Suitable acids for use in the preparation of pharmaceutically acceptablesalts include, but are not limited to, acetic acid, 2,2-dichloroaceticacid, acylated amino acids, adipic acid, alginic acid, ascorbic acid,L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, boric acid, (+)-camphoric acid, camphorsulfonic acid,(+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylicacid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid,D-glucuronic acid, L-glutamic acid, α-oxo-glutaric acid, glycolic acid,hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid,(+)-L-lactic acid, (+/−)-DL-lactic acid, lactobionic acid, lauric acid,maleic acid, (−)-L-malic acid, malonic acid, (+/−)-DL-mandelic acid,methanesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinicacid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid,pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid,saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid,stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaricacid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, andvaleric acid.

Suitable bases for use in the preparation of pharmaceutically acceptablesalts, including, but not limited to, inorganic bases, such as magnesiumhydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, orsodium hydroxide; and organic bases, such as primary, secondary,tertiary, and quaternary, aliphatic and aromatic amines, includingL-arginine, benethamine, benzathine, choline, deanol, diethanolamine,diethylamine, dimethylamine, dipropylamine, diisopropylamine,2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine,isopropylamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine,morpholine, 4-(2-hydroxyethyl)-morpholine, methylamine, piperidine,piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine,pyridine, quinuclidine, quinoline, isoquinoline, secondary amines,triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

The disclosure provides that compounds disclosed herein can have prodrugforms. Prodrugs of the compounds are useful in the methods of thisdisclosure. Any compound that will be converted in vivo to provide abiologically, pharmaceutically or therapeutically active form of acompound of the disclosure is a prodrug. Various examples and forms ofprodrugs are well known in the art. Examples of prodrugs are found,inter alia, in Design of Prodrugs, edited by H. Bundgaard, (Elsevier,1985), Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K.Widder, et al. (Academic Press, 1985); A Textbook of Drug Design andDevelopment, edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5,“Design and Application of Prodrugs,” by H. Bundgaard, at pp. 113-191,1991); H. Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38(1992); H. Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol.77, p. 285 (1988); and Nogrady (1985) Medicinal Chemistry A BiochemicalApproach, Oxford University Press, New York, pages 388-392).

Prodrugs of compounds disclosed herein can be prepared by methods knownto one of skill in the art and routine modifications thereof, and/orprocedures found in U.S. Pat No. 8,293,786, and references cited thereinand routine modifications made thereof.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy to administer by a syringe. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound, e.g. a compound disclosed herein, in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, themethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

In a particular embodiment, one or more compounds of the disclosure areprepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations should be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to cells with monoclonal antibodies) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound used in the method of thedisclosure, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC₅₀ (e.g., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

Compositions and formulations of one or more compounds disclosed hereincan be used in combination with other active agents to treat a disorderor disease in a subject.

It should be understood that the administration of an additionaltherapeutic agent with a compound of the disclosure encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, administration of an additional therapeuticagent in combination with a compound disclosed herein also encompassesuse of each type of therapeutic agent in a sequential manner. In eithercase, the treatment regimen will provide beneficial effects of the drugcombination in treating the disorders described herein.

In a further embodiment, the compounds disclosed herein can be combinedwith one or more class of therapeutic agents, including, but not limitedto, alkylating agents, cancer immunotherapy monoclonal antibodies,anti-metabolites, mitotic inhibitors, anti-tumor antibiotics,topoisomerase inhibitors, photosensitizers, tyrosine kinase inhibitors,anti-cancer agents, chemotherapeutic agents, anti-migraine treatments,anti-tussives, mucolytics, decongestants, anti-allergic non-steroidals,expectorants, anti-histamine treatments, anti-retroviral agents, CYP3Ainhibitors, CYP3A inducers, protease inhibitors, adrenergic agonists,anti-cholinergics, mast cell stabilizers, xanthines, leukotrieneantagonists, glucocorticoid treatments, antibacterial agents, antifungalagents, sepsis treatments, steroidals, local or general anesthetics,NSAIDS, NRIs, DARIs, SNRIs, sedatives, NDRIs, SNDRIs, monoamine oxidaseinhibitors, hypothalamic phoshpholipids, anti-emetics, ECE inhibitors,opioids, thromboxane receptor antagonists, potassium channel openers,thrombin inhibitors, growth factor inhibitors, anti-platelet agents,P2Y(AC) antagonists, anti-coagulants, low molecular weight heparins,Factor VIa inhibitors, Factor Xa inhibitors, renin inhibitors, NEPinhibitors, vasopepsidase inhibitors, squalene synthetase inhibitors,anti-atherosclerotic agents, MTP inhibitors, calcium channel blockers,potassium channel activators, alpha-muscarinic agents, beta-muscarinicagents, anti-arrhythmic agents, diuretics, thrombolytic agents,anti-diabetic agents, mineralocorticoid receptor antagonists, growthhormone secretagogues, aP2 inhibitors, phophodiesterase inhibitors,anti-inflammatories, anti-proliferatives, antibiotics, farnesyl-proteintransferase inhibitors, hormonal agents, plant-derived products,epipodophyllotoxins, taxanes, prenyl-protein transferase inhibitors,anti-TNF antibodies and soluble TNF receptors, Cyclooxygenase-2inhibitors, and miscellaneous agents.

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. Such kits cancomprise a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

For example, the container(s) can comprise one or more compoundsdescribed herein, optionally in a composition or in combination withanother agent as disclosed herein. The container(s) optionally have asterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). Such kits optionally comprise a compound with anidentifying description or label or instructions relating to its use inthe methods described herein.

A kit will typically comprise one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of a compound described herein. Non-limiting examplesof such materials include, but are not limited to, buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use. A set of instructions will alsotypically be included.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself, a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein. Theseother therapeutic agents may be used, for example, in the amountsindicated in the Physicians' Desk Reference (PDR) or as otherwisedetermined by one of ordinary skill in the art.

Traditionally, cartilage has been considered to be a static tissue, withlittle to no cellular turnover or capacity for repair following injury.However, detailed analysis of articular cartilage tissues from a varietyof species has identified a primitive stem/progenitor chondrocytepopulation located within the layer of cartilage closest to the jointspace, termed the superficial zone. These cells are capable ofproliferating and producing hyaline matrix, and mouse lineage tracingstudies show these cells maintain cartilage throughout life, but it isclear that they lack sufficient capacity to effect substantiveregeneration in most injury contexts. Moreover, there is a paucity ofinformation on how to positively identify these cells in humans andunderstand their biology. Finally, pro-inflammatory signaling that oftenaccompanies cartilage injury is a major inhibitor of proliferation whilealso driving apoptosis.

The pathogenesis of osteoarthritis (OA) often begins from an injury toarticular cartilage, which establishes chronic, low-grade inflammationmediated by interleukin-6/glycoprotein 130 (IL-6/gp130) and otherfactors that promote matrix degradation over time and eventualdestruction of cartilage. IL-6 signaling through IL-6R/gp130 suppresseschondrocyte proliferation, promotes mineralization in articularcartilage, downregulation of matrix proteins and increases expression ofmatrix-degrading proteases. Moreover, blockade of IL-6 in vivo in mousemodels of OA has been shown to be chondroprotective. Importantly, higherserum levels of IL-6 have been correlated with the development of OA inhumans, and a monoclonal antibody against IL-6R is currently in PhaseIII clinical trials for the treatment of hand OA (NCT02477059).Signaling downstream of IL-6/gp130 is mediated by multiple pathways,including signal transducer and activator of transcription 3 (STAT3).STAT3 has been demonstrated to have pleiotropic effects duringchondrogenesis and in articular chondrocytes. During chondrogenicdifferentiation of multipotent mesenchymal stem cells, IL-6/STAT3signaling promotes chondrocyte commitment and matrix production.Similarly, loss of STAT3 during limb formation results in increasedhypertrophy, premature ossification and decreases in expression of themaster regulator of chondrocyte identity SOX9. In contrast, in adultarticular chondrocytes inhibition of STAT3 downstream of IL-6 ischondroprotective, reducing the severity of OA-like pathology in a mousemodel. Together, these data indicate that IL-6/STAT3 signaling can drivematrix loss and development of OA in vivo in both mouse models andhumans.

Recent studies have shown that Bone Morphogenetic Protein receptor 1B(BMPR1B) marks superficial chondrocytes throughout human ontogeny andalso in rodent joints. As described herein these cells can also beidentified by their high level of IL-6 coreceptor gp130 expression andactivity. Based on the known role of IL-6/gp130 signaling in hypertrophyand OA pathogenesis, a small molecule screen was performed to identifypotential agents to manipulate gp130 signaling. These studies revealedRegulator of Cartilage Growth and Differentiation 423 (RCGD 423), asmall molecule modulator of gp130.

RCGD 423 has the general structure of Formula A:

wherein X is F or Br (see, e.g., WO/2016/138533, which is incorporatedherein by reference for all purposes).

In vitro studies demonstrated that RCGD 423 signals through gp130 andinteracts with the extracellular region of gp130. Further elucidation ofthe mechanism of RCGD 423 demonstrated that it promotes the formation ofactive, ligand-independent gp130 homodimers, thereby distinguishing itsactivity from IL-6 family cytokines; this was reflected in criticaldifferences in the downstream molecular events of IL-6 family cytokineand RCGD 423 stimulation. Moreover, RCGD 423 actively competes withsignaling by pro-inflammatory IL-6 family cytokines by sequesteringgp130 away from forming heterodimers with IL-6R. Finally, in a rat modelof OA, this molecule evidenced a remarkable ability to prevent cartilagedegeneration.

A close analog of RCGD 423 has been shown to stimulate hair cycle inmice through stabilization of MYC protein, thus validating the mechanismof action of this compound in a completely independent system. However,despite these positive results, increases in pSTAT3 and MYC levels maybe detrimental in a clinical OA pathology scenario, based on potentialpro-degenerative and oncogenic concerns, respectively.

RCGD 423 provided information regarding the specific regulatorypockets/clefts in gp130. Using this information, modeling and benchresearch led to the identification of small molecules that interactedwith the gp130 pockets/clefts. In particular, the research identified aplurality of molecules provided in Table 1. For example, one suchmolecule CX-011 (also referred to as “B8” herein) and related analogsare shown to be potent inhibitor of pro-catabolic signaling by IL-6family cytokines and which do not affect levels of pSTAT3 or MYCprotein.

In a particular embodiment, the disclosure provides for a compound thatinteracts with domain 2 of gp130 and which may lock gp130 into anon-permissive conformation and/or produce atypical gp130 homodimers,wherein the compound comprises the structure of Formula I, II or III:

wherein,X is

Y is

Z is

-   -   X¹ is S, CH, or NH;    -   X² is S, CH, or N;    -   X³ is CR² or S;    -   Y¹ is CR⁷ or N;    -   Y² is CR⁶ or N;    -   Y³ is CR⁵ or N;    -   Z¹ is O or CH;    -   Z² is O, N, NH or CH;    -   Z³ is CR⁹ or N;    -   Z⁴ is CR⁸ or N;    -   Z⁵ is N or CR¹⁴;    -   Z⁶ is N or CR¹³;    -   Z⁷ is N or CR¹²;    -   Z⁸ is N or CR¹¹;    -   Z⁹ is N or CR¹⁰;    -   v is 0 or 1;    -   R¹, R², R⁸ and R⁹ are independently selected from H, D, (C₁-C₃)        alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro, thiol, and        amino;    -   R³-R⁷ and R¹⁰-R¹⁴ are independently selected from H, D, (C₁-C₃)        alkyl, (C₁-C₃) alkenyl, halo, cyano, hydroxyl, nitro, thiol,        amino, OC (R¹⁵)₃, OCH(R¹⁵)₂, OCH2(R¹⁵),

wherein n is an integer from 1 to 5; and

-   -   R¹⁵ is independently H, halo, or a (C₁-C₃)alkyl.

In another embodiment, the disclosure provides for a compound thatinteracts with domain 2 of gp130 and which may lock gp130 into anon-permissive conformation and/or produce atypical gp130 homodimers,wherein the compound comprises the structure of Formula I, II or III:

wherein,

-   -   X is selected from the group consisting of

-   -   Y is

-   -   Z is

-   -   Y¹ is CR⁷ or N;    -   Y² is CR⁶ or N;    -   Y³ is CR⁵ or N;    -   Z¹ is O or CH;    -   Z² is O, N, NH or CH;    -   Z³ is CR⁹ or N;    -   Z⁴ is CR⁸ or N;    -   Z⁵ is N or CR¹⁴;    -   Z⁶ is N or CR¹³;    -   Z⁷ is N or CR¹²;    -   Z⁸ is N or CR¹¹;    -   Z⁹ is N or CR¹⁰;    -   v is 0 or 1;    -   R¹, R², R⁸ and R⁹ are independently selected from H, D, (C₁-C₃)        alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro, thiol, and        amino;    -   R³-R⁷ and R¹⁰-R¹⁴ are independently selected from H, D, (C₁-C₃)        alkyl, (C₁-C₃) alkenyl, halo, cyano, hydroxyl, nitro, thiol,        amino, OC (R¹⁵)₃, OCH(R¹⁵)₂, OCH2(R¹⁵),

wherein n is an integer from 1 to 5; and

-   -   R¹⁵ is independently H, halo, or a (C₁-C₃) alkyl.

In yet another embodiment, the disclosure provides for a compound thatinteracts with domain 2 of gp130 and which may lock gp130 into anon-permissive conformation and/or produce atypical gp130 homodimers,wherein the compound comprises the structure of Formula I, II or III:

wherein,

-   -   X is

-   -   Y is selected from the group consisting of:

-   -   Z is

-   -   X¹ is S, CH, or NH;    -   X² is S, CH, or N;    -   X³ is CR² or S;    -   Z¹ is O or CH;    -   Z² is O, N, NH or CH;    -   Z³ is CR⁹ or N;    -   Z⁴ is CR⁸ or N;    -   Z⁵ is N or CR¹⁴;    -   Z⁶ is N or CR¹³;    -   Z⁷ is N or CR¹²;    -   Z⁸ is N or CR¹¹;    -   Z⁹ is N or CR¹⁰;    -   v is 0 or 1;    -   R¹, R², R⁸ and R⁹ are independently selected from H, D, (C₁-C₃)        alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro, thiol, and        amino;    -   R³-R⁷ and R¹⁰-R¹⁴ are independently selected from H, D, (C₁-C₃)        alkyl, (C₁-C₃) alkenyl, halo, cyano, hydroxyl, nitro, thiol,        amino, OC (R¹⁵)₃, OCH(R¹⁵)₂, OCH2(R¹⁵),

wherein n is an integer from 1 to 5; and

-   -   R¹⁵ is independently H, halo, or a (C₁-C₃)alkyl.

In a further embodiment, the disclosure provides for a compound thatinteracts with domain 2 of gp130 and which can lock gp130 into anon-permissive conformation and/or produce atypical gp130 homodimers,wherein the compound comprises the structure of Formula I, II or III:

wherein,

-   -   X is

-   -   Y is

-   -   Z is selected from the group consisting of:

-   -   X¹ is S, CH, or NH;    -   X² is S, CH, or N;    -   X³ is CR² or S;    -   Y¹ is CR⁷ or N;    -   Y² is CR⁶ or N;    -   Y³ is CR⁵ or N;    -   v is 0 or 1;    -   R¹, R², R⁸ and R⁹ are independently selected from H, D,        (C¹-C₃)alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro,        thiol, and amino;    -   R³-R⁷ and R¹⁰-R¹⁴ are independently selected from H, D,        (C¹-C₃)alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro,        thiol, amino, OC(R¹⁵)₃, OCH(R¹⁵)₂, OCH2(R¹⁵),

wherein n is an integer from 1 to 5; and

-   -   R¹⁵ is independently H, halo, or a (C₁-C₃)alkyl.

In a particular embodiment, the disclosure provides for a compound thatinteracts with domain 2 of gp130 and which may lock gp130 into anon-permissive conformation and/or produce atypical gp130 homodimers,wherein the compound comprises the structure of Formula I, II or III:

wherein,

-   -   X is selected from the group consisting of:

-   -   Y is selected from the group consisting of:

-   -   Z is selected from the group consisting of:

-   -   v is 0 or 1;    -   R¹, R², R⁸ and R⁹ are independently selected from H, D,        (C¹-C₃)alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro,        thiol, and amino;    -   R³-R⁷ and R¹⁰-R¹⁴ are independently selected from H, D,        (C¹-C₃)alkyl, (C₁-C₃)alkenyl, halo, cyano, hydroxyl, nitro,        thiol, amino, OC(R¹⁵)₃, OCH(R¹⁵)₂, OCH₂(R¹⁵),

wherein n is an integer from 1 to 5; and

-   -   R¹⁵ is independently H, halo, or a (C₁-C₃)alkyl.

In a certain embodiment, the disclosure provides for a compound thatinteracts with domain 2 of gp130 and which locks gp130 into anon-permissive conformation and/or produce atypical gp130 homodimers,wherein the compound has a structure selected from the group consistingof:

Table 1 provides a list of molecules useful in the methods andcompositions of the disclosure.

TABLE 1 COMPOUND/MOLECULE NAME MW % purity

B8 285.32 99.1

423F 270.33 99.5

B801 285.32 99.4

B802 285.32 98.7

B803 285.32 99.5

B804 299.35 99.2

B805 295.36 99.5

B806 295.36 95.6

B807 284.34 99.7

B808 299.35 99.5

B809 285.32 98.9

B810 279.3 99.5

B811 299.35 97.9

B812 271.29 96.8

B813 337.30 99.5

B814 337.30 99.5

B815 285.32 99.3

B816 296.30 99.5

B817 296.30 98.6

B818 289.28 98.9

B819 289.28 98.7

B820 289.28 99.5

B821 272.28 96.5

B822 272.28 99.8

B823 272.28 95.3

B824 338.34 99.2

B825 245.26 99.5

B826 235.26 99.5

B827 285.32 99.0

B828 345.37 99.5

B829 345.37 99.5

B830 296.35 99.1

B831 296.35 98.8

B832 309.39 99.1

B833 309.39 98.3

B834 309.39 96.0

B835 325.39 97.9

B836 325.39 95.0

B837 325.39 98.8

B838 325.39 99.4

B839 289.34 99.5

B840 299.35

B841 345.37

B842 309.39

B843 295.36

B844 355.41

B845 295.36

B846 285.32

B847 251.3

B848 261.34

B849 279.3

CX-011 (B8) having the structure:

is predicted to bind gp130 in the same binding pocket as RCGD 423, andit is hypothesized that it stabilizes an inactive conformation. The invitro results, suggest a small molecule inhibitor of pro-inflammatory,pro-degenerative signaling mediated by IL-6 family cytokines throughgp130 would have great clinical importance. Although a biologic againstIL-6R is currently being tested as a therapeutic against OA, thistherapy does not block the effects of oncostatin M (OSM) and LIF, twoother IL-6 family members with pro-catabolic consequences on articularcartilage. Thus, small molecule gp130 inhibitor such as CX-011 (B8) andanalogs thereof are useful for post-traumatic OA and have a differentmethod of action. It is hypothesized that broad inhibition of IL-6family cytokine signaling will interrupt the pro-inflammatory,pro-degenerative environment present post-injury. In the experimentsdescribed herein and below, the half-maximal inhibitory concentration(IC₅₀) of CX-011 (B8) on gp130 in rat, dog and human articularchondrocytes in the presence of IL-6 and OSM are examined. Theanti-degenerative effects of CX-011 (B8) in two rat models of OA,focusing on proteoglycan loss, the levels of pro-catabolic enzymes andproliferation are examined. Together, the results demonstrate thefitness of CX-011 (B8) and analogs thereof to progress as a potentialtherapeutic candidate to combat OA.

The disclosure provides methods of treating inflammatory disorders,neoplasms, and cell proliferative disorders comprising contacting asubject or cell with any of the foregoing compounds. In otherembodiments, the disclosure provides methods of modulating theproduction or induction of inflammation and/or inflammatory cytokinescomprising contacting a cell or subject with a compound as describedherein. In certain embodiments, the cell is a chondrocyte.

The invention is illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

EXAMPLE

The gp130 co-receptor LIFR is expressed on a subset of superficialchondrocytes throughout human ontogeny. Based on these data, it ishypothesized that gp130 would also be expressed on these cells, and thiswas indeed the case (FIG. 1). Given the known (and mostly detrimentalroles) of IL-6 family cytokines on articular chondrocytes, a highthroughput screen was performed to identify small molecules that couldpotentially modulate gp130 signaling. In adult mice, IL-6/gp130signaling drives Co110a1 expression and hypertrophy, which has beenstrongly associated with OA progression; ˜170,000 compounds were thusscreened for their ability to prevent increases in Co110-mCherryfluorescence in mouse limb mesenchymal cells stimulated with BMP-4, astrong driver of hypertrophy (FIG. 2). One primary hit, RCGD 423,emerged after secondary screening as it could prevent increases inalkaline phosphatase, a marker of hypertrophy, in fetal articularchondrocytes (FIG. 2D).

To begin to address the mechanism of action of RCGD 423, adult pigchondrocytes were incubated with the drug, IL-6 family cytokines or thepro-catabolic cytokines TNF-α and IL-1β and measured levels of variousproteins downstream of these cytokines that are known to drive catabolicresponses (FIG. 3). These data revealed similarities among RCGD 423,IL-6 and LIF: all increased levels of active STAT3 and MYC proteins.However, while IL-6 and LIF activated the MAPK, AKT and NF-κB pathways,RCGD 423 did not evidence the same result (FIG. 4). This was confirmedby qPCR for the proteolytic enzymes ADAMTS4/5 and MMP13, which showedthat most tested cytokines including LIF and IL-6 resulted insignificant increases in the expression of these genes while RCGD 423did not (FIG. 5). Based on these data, it was hypothesized that RCGD 423interacted with gp130 to modify its signaling output. A small moleculeinhibitor of gp130 prevented the accumulation pSTAT3 and MYC proteinsfollowing incubation of adult pig articular chondrocytes with RCGD 423,suggesting that the compound acts near the apex of thegp130-JAK-STAT3-MYC signaling cascade (FIG. 6). Based on these results,it was further hypothesized that RCGD 423 is a direct agonist of gp130,inducing activation in the absence of ligand.

To address this hypothesis, the interaction of RCGD 423 with theextracellular domain of gp130 was modeled using Swissdock and theresults showed a putative high affinity binding site within domain 2 ofgp130 (FIG. 6). Four of the main amino acids interacting with thehalogenated ring of RCGD 423 (K151-R154) are of particular interest, asdeletion of these residues has been shown to cause irreversibleactivation of gp130 signaling by promoting the formation ofconstitutively active homodimers, driving a gene expression responsetypical of high STAT3 activity. These findings show that K151-R154 areimportant for inhibiting inappropriate activation of gp130. Of note,another gp130 deletion mutant identified and tested demonstrated thatloss of key residues in gp130 leads to unbridled gp130 signaling throughnon-canonical homodimers.

Based on this, it was hypothesized that RCGD 423 acts through gp130 bypromoting stable homodimers in the absence of ligand. To confirm thatRCGD 423 acts through gp130, the gp130 null cell line Ba/F3 cells wasused. Ba/F3 cells were transfected with either full-length gp130 or amutant gp130 lacking domain 2 (ΔD2), which contains the predictedbinding site for RCGD 423 (FIG. 7). These data demonstrated thatfull-length gp130 is required for the activity of RCGD 423. To verifythe specificity of RCGD 423 in vivo, the compound was injected into ratknees in the presence of the small molecule gp130 inhibitor SC144 or ablocking antibody specific for domain 2 of gp130. Inclusion of eitherantagonist completely inhibited signaling downstream of RCGD 423 (FIG.8). The ability of RCGD 423 to act via inducing formation of gp130homodimers was then examined by expressing tagged versions of gp130 in293T cells. In this system, only homodimeric complexes can contain bothtagged versions of gp130 (FIG. 9). The results demonstrated that only inthe presence of RCGD 423 could gp130 homodimers be detected. Together,these results demonstrated that RCGD 423 is a direct gp130 agonist thatacts by promoting formation of active gp130 homodimers in the absence ofligand.

Based on this mechanism of action, it was hypothesized that RCGD 423could potentially inhibit signaling by IL-6 family cytokines due to therequirement for gp130 to interact with ligand/receptor complexes. Thisproperty would be of great interest due to the pro-inflammatory andpro-catabolic effects of these cytokines in the joint space. Incubationof human adult articular chondrocytes with RCGD 423 in combination withOSM or IL-6 significantly reduced increases in NF-κB protein, a majorregulator of inflammation and catabolism (FIG. 10A). To assess whetherRCGD 423 could directly compete for gp130 occupancy, human adultarticular chondrocytes were transfected with tagged gp130 and thenpulled down associated proteins±RCGD 423 and/or IL-6. These resultsdemonstrated that in the presence of RCGD 423, gp130-IL-6R complexeswere significantly reduced (FIG. 10B). These results were expanded todetermine the dissociation constant (K_(d)), a measure of the affinitybetween gp130 and its binding partners OSMR and IL-6R, in the presenceof RCGD 423 and either OSM (oncostatin M) or IL-6 (FIG. 11). Theseresults demonstrated a strong ability of the compound to interfere withligand-mediated gp130 heterodimerization. Taken together, these dataidentify an additional mechanism of action by which RCGD 423 couldevidence disease-modifying activity.

Based on these results, it was hypothesized that RCGD 423 could evidencedisease-modifying activity in a rat model of degenerativeosteoarthritis. In this system, 50% of the meniscus is surgicallyremoved, promoting joint instability similar to what is commonlyobserved in human OA and degeneration of articular cartilage in 3-6weeks (FIGS. 12A and B). RCGD 423 loaded onto PLGA(poly(lactic-co-glycolic acid)) microspheres for slow release, ormicrospheres alone, were injected into the joint space at the time ofsurgery and 3 weeks later. This dosing interval was based on off-loadingstudies which showed continuous released of RCGD 423 from PLGAmicrospheres over 3-4 weeks in culture (data not shown). After 6 weeks,knees treated with RCGD 423 evidenced highly significantly lower OARSIscores, a histological measure of OA that increases with worseningcartilage loss and joint damage (FIGS. 12C and D). Moreover, RCGD 423decreased the expression of proteolytic enzymes that promote cartilageloss following injury (FIG. 12E). In addition, the compound alsoinhibited both the formation and maturity of osteophytes in response tojoint injury (FIG. 13). These results provide conclusive data that RCGD423 prevents cartilage degeneration in vivo.

Pathogenesis of OA is a process driven by the entire joint, not justchondrocytes; both the synovium and infiltrating immune cells have beendemonstrated to secrete not only IL-6, but other pro-inflammatorycytokines as well. Accordingly, the ability of RCGD 423 to suppresssignaling downstream of IL-6 and OSM in synoviocytes and peripheralblood mononuclear cells was assessed (FIG. 14). These resultsdemonstrate a potent suppression of signaling in these cell types whichpromote cartilage loss in injured joints, defining another potentialmechanism by which RCGD 423 could interrupt the pro-degenerative cycle.

Based on these data, a small molecule modulator of gp130 signaling wasidentified that elicited a distinct molecular profile from IL-6 familycytokines: high levels of pSTAT3 and MYC, low induction of NF-κB andsignaling through ligand-independent gp130 homodimers. Functionally,these effects result in a blockade of pro-inflammatory signalingdownstream of IL-6 family cytokines, including increases inmatrix-degrading enzymes; it was hypothesized that these are the causeof the anti-degenerative effects of RCGD 423 in vivo.

Based on these data, derivatives of RCGD 423 were developed for severalreasons. Firstly, RCGD 423 was a hit from a library; this made the depthof the discovery surrounding the technology less comprehensive.Additionally, the physical properties of RCGD 423 (solubility, potency,functional groups) could be improved. Significant gains in these areascould potentially result in a first-in-class injectable for thetreatment of post-traumatic OA. Moreover, the potential risks associatedwith promoting MYC stabilization and proliferation in gp130⁺ cells aswell as large increases in active STAT3 protein was of concern.Therefore, a cohort of structural variants based on the RCGD 423scaffold were generated that were predicted to bind in the same pocketof gp130 and possess improved physical properties. These compounds werethen screened in adult pig articular chondrocytes and cartilageexplants, seeking compounds that could inhibit the catabolic effects ofOSM but not drive increases in MYC and pSTAT3 protein levels. Oneanalog, CX-011 (B8), stood out as it almost completely blocked thegeneration of aggrecan and collagen neoepitopes, indicative of stronganti-catabolic potential, and did not increase levels of pSTAT3 and MYC(FIGS. 15A and B). Moreover, CX-011 (B8) reduced the induction ofproteolytic gene expression, further supporting the neoepitope data(FIG. 15C). Importantly, unlike some other analogs tested, CX-011 (B8)did not strongly effect basal levels of MYC and pSTAT3 proteins, whichmay play important roles in maintaining chondrocyte homeostasis (FIG.15A). The interaction between CX-011 (B8) and gp130 was modeled, whichsuggested and strong binding of the compound to the same binding siteoccupied by RCGD 423. Together, these preliminary data identify CX-011(B8) as a highly anti-inflammatory, anti-catabolic alternative to RCGD423, with increased “drug-like” properties and reduced potentialmolecular liabilities.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of treating osteoarthritis comprisingcontacting a human subject in need thereof with a therapeuticallyeffective amount of a pharmaceutical composition comprising a suspensionof compound 805 in a pharmaceutically acceptable carrier formulated forinjection:


2. The method of claim 1, wherein the pharmaceutical composition furthercomprises hyaluronic acid.
 3. The method of claim 1, further comprisingcontacting the subject with an additional therapeutic compound selectedfrom the group consisting of glucocorticoid treatments, steroidals,local or general anesthetics, NSAIDS, low molecular weight heparins,anti-inflammatories, anti-TNF antibodies and soluble TNF receptors andcyclooxygenase-2 inhibitors.
 4. The method of claim 1, wherein thecomposition is effective to treat osteoarthritis at less than about 10mg/kg of body weight.
 5. The method of claim 1, wherein the compositionis sterilized by filtration.
 6. The method of claim 1, wherein thepharmaceutical composition is formulated for injection into the knee orhip.
 7. The method of claim 1, wherein the osteoarthritis affects thesynovium by promoting infiltration and activation of immune cells.